AU7166500A - New nucleotide sequences which code for the pfk gene - Google Patents

Description

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AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name and Degussa-Huls Aktiengesellschaft Address DE-60287Frankfurt am Main of Applicant: Germany Actual Bettina Mockel and Walter Pfefferle Inventor(s): Address for Spruson Ferguson Service: St Martins Tower 31 Market Street Sydney NSW 2000 Invention Title: New Nucleotide Sequences which Code for the pfk Gene The following statement is a full description of this invention, including the best method of performing it known to me/us:- **°ooo• *ooooo* 5845c 990179 BT AL 1 New nucleotide sequences which code for the pfk gene The invention provides nucleotide sequences which code for the pfk gene and a process for the fermentative preparation of amino acids, in particular L-lysine, using coryneform bacteria in which the pfk gene is enhanced.

Prior art Amino acids, in particular L-lysine, are used in human medicine and in the pharmaceuticals industry, but in particular in animal nutrition.

It is known that amino acids are prepared by fermentation from strains of coryneform bacteria, in particular Corynebacterium glutamicum. Because of their great importance, work is constantly being undertaken to improve the preparation processes. Improvements to the processes can relate to fermentation measures, such as e. g. stirring and supply of oxygen, or the composition of the nutrient media, such as e. g. the sugar concentration during the fermentation, or the working up to the product form by e. g. ion exchange chromatography, or the intrinsic output properties of the microorganism itself.

Methods of mutagenesis, selection and mutant selection are used to improve the output properties of these -microorganisms. Strains which are resistant to antimetabolites, such as e. g. the lysine analogue S-(2aminoethyl)-cysteine, or are auxotrophic for metabolites of regulatory importance and produce L-amino acids, such as e. g. L-lysine, are obtained in this manner.

Methods of the recombinant DNA technique have also been employed for some years for improving the strain of Corynebacterium strains which produce amino acids, by amplifying individual amino acid biosynthesis genes and investigating the effect on the amino acid production.

990179 BT AL 2 Review articles in this context are to be found, inter alia, in Kinoshita ("Glutamic Acid Bacteria", in: Biology of Industrial Microorganisms, Demain and Solomon (Eds.), Benjamin Cummings, London, UK, 1985, 115-142), Hilliger (BioTec 2, 40-44 (1991)), Eggeling (Amino Acids 6:261-272 (1994)), Jetten and Sinskey (Critical Reviews in Biotechnology 15, 73-103 (1995)) and Sahm et al. (Annuals of the New York Academy of Science 782, 25-39 (1996)).

2 0 *oo* 990179 BT AL 3 Object of the invention The inventors had the object of providing new measures for improved fermentative preparation of amino acids, in particular L-lysine.

Description of the invention Amino acids, in particular L-lysine, are used in human medicine, in the pharmaceuticals industry and in particular in animal nutrition. There is therefore a general interest in providing new improved processes for the preparation of amino acids, in particular L-lysine.

When L-lysine or lysine are mentioned in the following, not only the base but also the salts, such as e. g. lysine monohydrochloride or lysine sulfate, are also meant.

The invention provides an isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence chosen from the group consisting of a) polynucleotide which is identical to the extent of at least 70 to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of 20 SEQ ID No. 2, b) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70% to the amino acid sequence of SEQ ID No.2, 25 c) polynucleotide which is complementary to the polynucleotides of a) or and d) polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequence of b) or The invention also provides the polynucleotide this preferably being a DNA which is capable of replication, comprising: the nucleotide sequence shown in SEQ ID No. 1, or (ii) at least one sequence which corresponds to sequence within the range of the degeneration f the genetic code, or (iii) at least one sequence which hybridizes with the sequence complementary to sequence or and optionally (iv) sense mutations of neutral function in The invention also provides o0 a polynucleotide comprising the nucleotide sequence as shown in SEQ ID No. 1, a polynucleotide which codes for a polypeptide which comprises the amino acid sequence as shown in SEQ ID No. 2, a vector containing a polynucleotide of the invention in particular a shuttle vector or plasmid vector and coryneform bacteria serving as the host cell, which contain the vector.

The invention also provides polynucleotides which substantially comprise a polynucleotide sequence, which are obtainable by screening by means of hybridization of a corresponding gene library, which comprises the complete gene with the polynucleotide sequence corresponding to SEQ ID No. 1, with a probe which comprises the sequence of i 20 the polynucleotide mentioned, according to SEQ ID No. 1 or a fragment thereof, and isolation of the DNA sequence mentioned.

eeoeo* [I:\DayLib\LIBC]8643.doc:kw- 990179 BT AL Polynucleotide sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA, in order to isolate, in the full length, cDNA which code for phosphofructokinase and to isolate those cDNA or genes which have a high similarity of sequence with that of the phosphofructokinase gene.

Polynucleotide sequences according to the invention are furthermore suitable as primers for the preparation of DNA of genes which code for phosphofructokinase by the polymerase chain reaction (PCR).

Such oligonucleotides which serve as probes or primers comprise at least 30, preferably at least 20, very particularly preferably at least 15 successive nucleotides.

Oligonucleotides which have a length of at least 40 or nucleotides are also suitable.

"Isolated" means separated out of its natural environment.

"Polynucleotide" in general relates to polyribonucleotides and polydeoxyribonucleotides, it being possible for these to be non-modified RNA -or DNA or modified RNA or DNA.

20 "Polypeptides" is understood as meaning peptides or proteins which comprise two or more amino acids bonded via peptide bonds.

The polypeptides according to the invention include a polypeptide according to SEQ ID No. 2, in particular those with the biological activity of phosphofructokinase, and also those which are identical to the extent of at least 70 to the polypeptide according to SEQ ID No. 2, and preferably are identical to the extent of 80% and in particular to the extent of 90 to 95 to the polypeptide 30 according to SEQ ID no. 2 and have the activity mentioned.

The invention also provides a process for the fermentative preparation of amino acids, in particular L-lysine, using 990179 BT AL 6 coryneform bacteria which in particular already produce an amino acid, and in which the nucleotide sequences which code for the pfk gene are enhanced, in particular overexpressed.

The term "enhancement" in this connection describes the increase in the intracellular activity of one or more enzymes in a microorganism which are coded by the corresponding DNA, for example by increasing the number of copies of the gene or genes, using a potent promoter or using a gene which codes for a corresponding enzyme having a high activity, and optionally combining these measures.

The microorganisms which the present invention provides can prepare L-amino acids, in particular L-lysine, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. They can be representatives of coryneform bacteria, in particular of the genus Corynebacterium. Of the genus Corynebacterium, there may be mentioned in particular the species Corynebacterium glutamicum, which is known among experts 20 for its ability to produce L-amino acids.

Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are, for example, the known wild-type strains Corynebacterium glutamicum ATCC13032 25 Corynebacterium acetoglutamicum ATCC15806 Corynebacterium acetoacidophilum ATCC13870 Corynebacterium thermoaminogenes FERM BP-1539 Corynebacterium melassecola ATCC17965 Brevibacterium flavum ATCC14067 30 Brevibacterium lactofermentum ATCC13869 and Brevibacterium divaricatum ATCC14020 and L-lysine-producing mutants or strains prepared therefrom, such as, for example I .1 990179 BT AL 7 Corynebacterium glutamicum FERM-P 1709 Brevibacterium flavum FERM-P 1708 Brevibacterium lactofermentum FERM-P 1712 Corynebacterium glutamicum FERM-P 6463 Corynebacterium glutamicum FERM-P 6464 and Corynebacterium glutamicum DSM5715.

The inventors have succeeded in isolating the new pfk gene of C. glutamicum which codes for the enzyme phosphofructokinase.

To isolate the pfk gene or also other genes of C.

glutamicum, a gene library of this microorganism is first set up in E. coli. The setting up of gene libraries is described in generally known textbooks and handbooks. The textbook by Winnacker: Gene und Klone, Eine Einfuhrung in die Gentechnologie [Genes and Clones, An Introduction to Genetic Engineering] (Verlag Chemie, Weinheim, Germany, 1990) or the handbook by Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) may be mentioned as an example. A well-known 20 gene library is that of the E. coli K-12 strain W3110 set up in vectors by Kohara et al. (Cell 50, 495-508 (1987)).

Bathe et al. (Molecular and General Genetics, 252:255-265, 1996) describe a gene library of C. glutamicum ATCC13032, which was set up with the aid of the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84:2160-2164) in the E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575). Bbrmann et al. (Molecular Microbiology 6(3), 317-326)) (1992)) in turn describe a gene library of C.

glutamicum ATCC13032 using the cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)). To prepare a gene library of C. glutamicum in E. coli it is also possible to use plasmids such as pBR322 (Bolivar, Life Sciences, 807-818 (1979)) or pUC9 (Vieira et al., 1982, Gene, 19:259- 268). Suitable hosts are, in particular, those E. coli 990179 BT AL 8 strains which are restriction- and recombination-defective.

An example of these is the strain DH5Smcr, which has been described by Grant et al. (Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649). The long DNA fragments cloned with the aid of cosmids can then in turn be subcloned in the usual vector suitable for sequencing and then sequenced, as is described e. g. by Sanger et al.

(Proceedings of the National Academy of Sciences of the United States of America, 74:5463-5467, 1977).

The new DNA sequence of C. glutamicum which codes for the pfk gene and which, as SEQ ID No. 1, is a constituent of the present invention, was obtained in this manner. The amino acid sequence of the corresponding protein has furthermore been derived from the present DNA sequence by the methods described above. The resulting amino acid sequence of the pfk gene product is shown in SEQ ID No. 2.

Coding DNA sequences which result from SEQ ID NO. 1 by the degeneracy of the genetic code are also a constituent of the invention. In the same way, DNA sequences which hybridize with SEQ ID NO. 1 or parts of SEQ ID NO. 1 are a constituent of the invention. Conservative amino acid exchanges, such as e. g. exchange of glycine for alanine or of aspartic acid for glutamic acid in proteins, are furthermore known among experts as "sense mutations" which 25 do not lead to a fundamental change in the activity of the protein, i.e. are of neutral function. It is furthermore known that changes on the N and/or C terminus of a protein cannot substantially impair or can even stabilize the function thereof. Information in this context can be found by the expert, inter alia, in Ben-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)), in O'Regan et al. (Gene 77:237-251 (1989)), in Sahin-Toth et al. (Protein Sciences 3:240-247 (1994)), in Hochuli et al. (Bio/Technology 6:1321-1325 (1988)) and in known textbooks of genetics and molecular biology. Amino acid sequences which result in a corresponding manner from SEQ ID No. 2 are also a constituent of the invention.

In the same way, DNA sequences which hybridize with SEQ ID No. 1 or parts of SEQ ID No. 1 are a constituent of the invention. Finally, DNA sequences which are prepared by the polymerase chain reaction (PCR) using primers which result from SEQ ID No. 1 are a constituent of the invention. Such oligonucleotides typically have a length of at least 15 nucleotides.

Instructions for identifying DNA sequences by means of hybridization can be found by the expert, inter alia, in the handbook "The DIG System Users Guide for Filter Hybridization" from Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al. (International Journal of Systematic Bacteriology (1991) 41: 255-260).

Instructions for amplification of DNA sequences with the aid of the polymerase chain reaction (PCR) can be found by the expert, inter alia, in the handbook by Gait: Oligonucleotide synthesis: a practical approach (IRL Press, Oxford, UK, 1984) and in Newton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).

The inventors have found that coryneform bacteria produce amino acids, in particular L-lysine, in an improved manner after over-expression of the pfk gene.

To achieve an over-expression, the number of copies of the corresponding genes can be increased, or the promoter and regulation region or the ribosome binding site I,"g 20 upstream of the structural gene can be mutated. Expression cassettes which are incorporated upstream of the structural gene act in the same way. By inducible promoters, it is additionally possible to increase the expression in the course of fermentative L-lysine production. The expression is likewise improved by measures to prolong the life of the m-RNA. Furthermore, the enzyme activity is also increased by Sess l

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Oo•,ii [I:\DayLib\LIBC]08643.doc:kww 990179 BT AL preventing the degradation of the enzyme protein. The genes or gene constructions can either be present in plasmids with a varying number of copies, or can be integrated and amplified in the chromosome. Alternatively, an overexpression of the genes in question can furthermore be achieved by changing the composition of the media and the culture procedure.

Instructions in this context can be found by the expert, inter alia, in Martin et al. (Bio/Technology 5, 137-146 (1987)), in Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), in European Patent Specification EPS 0 472 869, in US Patent 4,601,893, in Schwarzer and Puhler (Bio/Technology 9, 84-87 (1991), in Reinscheid et al. (Applied and Environmental Microbiology 126-132 (1994)), in LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), in Patent Application WO 96/15246, in Malumbres et al. (Gene 134, 15 24 (1993)), in Japanese Laid-Open Specification 229891, in Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), in Makrides (Microbiological Reviews 60:512-538 (1996)) and in known textbooks of genetics and molecular biology.

By way of example, the pfk gene according to the invention 25 was over-expressed with the aid of plasmids.

Suitable plasmids are those which are replicated in coryneform bacteria. Numerous known plasmid vectors, such as e. g. pZl (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKExl (Eikmanns et al., 30 Gene 102:93-98 (1991)) or pHS2-1 (Sonnen et al., Gene 107:69-74 (1991)) are based on the cryptic plasmids pHM1519, pBL1 or pGA1. Other plasmid vectors, such as e. g.

those based on pCG4 (US-A 4,489,160) or pNG2 (Serwold-Davis eeeeoe e 990179 BT AL 11 et al., FEMS Microbiology Letters 66, 119-124 (1990)), or pAG1 (US-A 5,158,891) can be used in the same manner.

Plasmid vectors which are furthermore suitable are those with the aid of which the process of gene amplification by integration into the chromosome can be used, as has been described, for example, by Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) for duplication or amplification of the hom-thrB operon. In this method, the complete gene is cloned in a plasmid vector which can replicate in a host (typically E. coli), but not in C. glutamicum. Possible vectors are, for example, pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983)), pK18mob or pKl9mob (Schafer et al., Gene 145, 69- 73 (1994)), pGEM-T (Promega corporation, Madison, WI, USA), pCR2.1-TOPO (Shuman (1994). Journal of Biological Chemistry 269:32678-84; US-A 5,487,993), pCR®Blunt (Invitrogen, Groningen, Holland; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)) or pEM1 (Schrumpf et al, 1991, Journal of Bacteriology 173:4510-4516). The plasmid vector which contains the gene to be amplified is then transferred into the desired strain of C. glutamicum by conjugation or transformation. The method of conjugation-is described, for example, by Schafer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Methods for S 25 transformation are described, for example, by Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)). After homologous recombination by means of a "cross over" event, the resulting strain contains at least two copies of the gene in question.

In addition, it may be advantageous for the production of amino acids, in particular L-lysine, to enhance or overexpress one or more enzymes of the particular biosynthesis 0 990179 BT AL 12 pathway, of glycolysis, of anaplerosis, of the citric acid cycle or of amino acid export, in addition to the pfk gene.

Thus, for example, for the preparation of L-lysine, one or more genes chosen from the group consisting of the dapA gene which codes for dihydrodipicolinate synthase (EP-B 0 197 335), or the gap gene which codes for glyceraldehyde 3-phosphate dehydrogenase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086), or the tpi gene which codes for triose phosphate isomerase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086), or the pgk gene which codes for 3-phosphoglycerate kinase(Eikmanns (1992), Journal of Bacteriology 174:6076- 6086), or the pyc gene which codes for pyruvate carboxylase(Eikmanns (1992), Journal of Bacteriology 174:6076-6086), or 2* the lysE gene which codes for lysine export 20 (DE-A-195 48 222) can be over-expressed at the same time.

For the production of amino acids, in particular L-lysine, it may furthermore be advantageous to attenuate, in addition to the pfk gene, 25 the pck gene which codes for phosphoenol pyruvate carboxykinase (DE 199 50 409.1, DSM 13047) and/or the pgi gene which codes for glucose 6-phosphate isomerase (US 09/396,478, DSM 12969) at the same time.

In addition to over-expression of the pfk gene it may furthermore be advantageous, for the production of amino acids, in particular L-lysine, to eliminate undesirable side reactions, (Nakayama: "Breeding of Amino Acid Producing Micro-organisms", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek Academic Press, London, UK, 1982).

The microorganisms prepared according to the invention can be cultured continuously or discontinuously in the batch process (batch culture) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) for the purpose of production of amino acids, in particular L-lysine. A summary of known culture methods is described in the textbook by Chmiel (Bioprozesstechnik 1. Einfiihrung in die Bioverfahrenstechnik [Bioprocess Technology 1. Introduction to Bioprocess Technology (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioraktoren und periphere Einrichtungen [Bioreactors and Peripheral Equipment] (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

The culture medium to be used must meet the requirements of the particular strains in a suitable manner. Descriptions of culture media for various microorganisms are contained in the handbook "Manual of Methods of General Bacteriology" of the American Society for Bacteriology (Washington USA, 1981). Sugars and carbohydrates such as e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as e.g. soya oil, sunflower oil, groundnut oil and coconut fat, fatty acids, such as e.g. palmatic acid, stearic acid and linoleic acid, alcohols, such as e.g. glycerol and ethanol, and organic acids, such as e.g. acetic acid, can be used as the source of carbon. These substances can be o *555 [I:\DayLib\LIBC]08643.doc:kww 990179 BT AL 14 used individually or as a mixture. Organic nitrogencontaining compounds, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya bean flour and urea, or inorganic compounds, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, can be used as the source of nitrogen. The sources of nitrogen can be used individually or as a mixture. Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the source of phosphorus. The culture medium must furthermore comprise salts of metals, such as e. g. magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth substances, such as amino acids and vitamins, can be employed in addition to the abovementioned substances. Suitable precursors can moreover be added to the culture medium. The starting substances mentioned can be added to the culture in the form of a single batch, or can be fed in during the culture in a suitable manner.

Basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acid compounds, such as phosphoric acid or sulfuric acid, can be employed *in a suitable manner to control the pH. Antifoams, such as e. g. fatty acid polyglycol esters, can be employed to control the development of foam. Suitable substances having a selective action, such as e. g. antibiotics, can be added to the medium to maintain the stability of plasmids. To *0000 maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, such as e. g. air, are introduced into the culture. The temperature of the culture is usually 20'C to 45 0 C, and preferably 25 0 C to 40 0 C. Culturing is continued until a maximum of lysine has formed. This target is usually reached within 10 hours to 160 hours.

The analysis of L-lysine can be carried out by anion exchange chromatography with subsequent ninhydrin 990179 BT AL derivatization, as described by Spackman et al. (Analytical Chemistry, 30, (1958), 1190).

The process according to the invention is used for the fermentative preparation of amino acids, in particular Llysine.

990179 BT AL 16 Examples The present invention is explained in more detail in the following with the aid of embodiment examples.

Example 1 Preparation of a genomic cosmid gene library from Corynebacterium glutamicum ATCC 13032 Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 was isolated as described by Tauch et al. (1995, Plasmid 33:168-179) and partly cleaved with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product Description Sau3AI, Code no. 27-0913-02). The DNA fragments were dephosphorylated with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, Product Description SAP, Code no. 1758250). The DNA of the cosmid vector SuperCosl (Wahl et al. (1987) Proceedings of the National Academy of Sciences USA 84:2160-2164), obtained from the company Stratagene (La Jolla, USA, Product Description SuperCosl Cosmid Vektor Kit, Code no. 251301) 20 was cleaved with the restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany, Product Description XbaI, Code no. 27-0948-02) and likewise dephosphorylated with shrimp alkaline phosphatase. The cosmid DNA was then cleaved with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Code no. 27-0868-04). The cosmid DNA treated in this manner e* was mixed with the treated ATCC13032 DNA and the batch was treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4-DNA-Ligase, Code no.27- 30 0870-04). The ligation mixture was then packed in phages with the aid of Gigapack II XL Packing Extracts (Stratagene, La Jolla, USA, Product Description Gigapack II XL Packing Extract, Code no. 200217). For infection of the 990179 BT AL 17 E. coli strain NM554 (Raleigh et al. 1988, Nucleic Acid Research 16:1563-1575) the cells were taken up in mM MgSO 4 and mixed with an aliquot of the phage suspension. The infection and titering of the cosmid library were carried out as described by Sambrook et al.

(1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor), the cells being plated out on LB agar (Lennox, 1955, Virology, 1:190) with 100 pg/ml ampicillin. After incubation overnight at 37 0 C, recombinant individual clones were selected.

Example 2 Isolation and sequencing of the pfk gene The cosmid DNA of an individual colony was isolated with the Qiaprep Spin Miniprep Kit (Product No. 271.06, Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions and partly cleaved with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product Description Sau3AI, Product No. 27-0913-02). The DNA fragments were dephosphorylated with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, Product Description SAP, Product No. 1758250).

After separation by gel electrophoresis, the cosmid fragments in the size range of 1500 to 2000 bp were *:*isolated with the QiaExII Gel Extraction Kit (Product No.

25 20021, Qiagen, Hilden, Germany). The DNA of the sequencing vector pZero-1, obtained from the company Invitrogen (Groningen, The Netherlands, Product Description Zero Background Cloning Kit, Product No. K2500-01) was cleaved with the restriction enzyme BamHI (Amersham Pharmacia, 30 Freiburg, Germany, Product Description BamHI, Product No.

27-0868-04). The ligation of the cosmid fragments in the sequencing vector pZero-1 was carried out as described by Sambrook et al. (1989, Molecular Cloning: A laboratory o Manual, Cold Spring Harbor), the DNA mixture being 990179 BT AL 18 incubated overnight with T4 ligase (Pharmacia Biotech, Freiburg, Germany). This ligation mixture was then electroporated (Tauch et al. 1994, FEMS Microbiol Letters, 123:343-7) into the E. coli strain DH5aMCR (Grant, 1990, Proceedings of the National Academy of Sciences U.S.A., 87:4645-4649) and plated out on LB agar (Lennox, 1955, Virology, 1:190) with 50 pg/ml zeocin. The plasmid preparation of the recombinant clones was carried out with Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany). The sequencing was carried out by the dideoxy chain termination method of Sanger et al. (1977, Proceedings of the National Academy of Sciences U.S.A., 74:5463-5467) with modifications according to Zimmermann et al. (1990, Nucleic Acids Research, 18:1067). The "RR dRhodamin Terminator Cycle Sequencing Kit" from PE Applied Biosystems (Product No. 403044, Weiterstadt, Germany) was used. The separation by gel electrophoresis and analysis of the sequencing reaction were carried out in a "Rotiphoresis NF Acrylamide/Bisacrylamide" Gel (29:1) (Product No.

A124.1, Roth, Karlsruhe, Germany) with the "ABI Prism 377" sequencer from PE Applied Biosystems (Weiterstadt, Germany).

The raw sequence data obtained were then processed using the Staden program package (1986, Nucleic Acids Research, 14:217-231) version 97-0. The individual sequences of the pZerol derivatives were assembled to a continuous contig.

The computer-assisted coding region analysis [sic] were prepared with the XNIP program (Staden, 1986, Nucleic Acids Research, 14:217-231). Further analyses were carried out with the "BLAST search program" (Altschul et al., 1997, Nucleic Acids Research, 25:3389-3402), against the nonredundant databank of the "National Center for Biotechnology Information" (NCBI, Bethesda, MD, USA).

The resulting nucleotide sequence is shown in SEQ ID No. 1.

S 35 Analysis of the nucleotide sequence showed an open reading 990179 BT AL 19 frame of 990 base pairs, which was designated the pfk gene.

The pfk gene codes for a protein of 330 amino acids.

Example 3 Preparation of the expression vector pZ-pfkex for enhancement of the pfk gene in Corynebacterium glutamicum 3.1. Cloning of the pfk gene From the strain ATCC 13032, chromosomal DNA was isolated by the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)). On the basis of the sequence of the pfk gene known for C. glutamicum from example 2, the following oligonucleotides were chosen for the polymerase chain reaction: pfk-ex: GAT CTA GAA TTC AAC TTT CAG GTG GTA ACC C 3' pfk-glp2: GAT CTA GTC GAC CGG ACA AGC GAG GAA TTA T 3' The primers described were synthesized by ARK Scientific GmbH Biosystems (Darmstadt, Germany). The primer pfk-ex contains the sequence for the cleavage site of the 20 restriction endonuclease EcoRI and the primer pfk-glp2 the cleavage site of the restriction endonuclease SalI, which are marked by underlining in the nucleotide sequence shown above. The PCR reaction was carried out by the standard PCR method of Innis et al. (PCR protocols. A Guide to Methods 25 and Applications, 1990, Academic Press) with Pwo-Polymerase from Roche Diagnostics GmbH (Mannheim, Germany). With the aid of the polymerase chain reaction, the primers allow amplification of a DNA fragment of approx. 1.05 kb in size, which carries the pfk gene from Corynebacterium glutamicum.

The product amplified in this way was tested electrophoretically in a 0.8% agarose gel.

990179 BT AL The PCR fragment obtained in this manner was cleaved completely with the restriction enzymes EcoRI and SalI. The pfk fragment approx. 1.05 kb in size was isolated from the agarose gel with the QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany).

3.2. Cloning of pfk in the vector pZ8-1 The E. coli C. glutamicum shuttle expression vector pZ8-1 (EP 0 375 889) was employed as the base vector for expression both in C. glutamicum and in E. coli. DNA of this plasmid was cleaved completely with the restriction enzymes EcoRI and SalI and then dephosphorylated with shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, Product Description SAP, Product No.

1758250). The pfk fragment isolated from the agarose gel in example 3.1 was mixed with the vector pZ8-1 prepared in this way and the batch was treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4-DNA-Ligase, Code no.27-0870-04).

The ligation batch was transformed in the E. coli strain DH5amcr (Hanahan, In: DNA cloning. A Practical Approach, Vol. I, IRL-Press, Oxford, Washington DC, USA). Selection of plasmid-carrying cells was made by plating out the transformation batch on LB agar (Lennox, 1955, Virology, 1:190) with 50 mg/l kanamycin. After incubation overnight 25 at 37°C, recombinant individual clones were selected.

Plasmid DNA was isolated from a transformant with the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions and investigated by restriction cleavage. The 30 resulting plasmid was called pZ-pfkex. It is shown in S* figure 1.

990179 BT AL 21 Example 4 Transformation of the strain DSM5715 with the plasmid pZpfkex The strain DSM5715 was transformed with the plasmid pZpfkex using the electroporation method described by Liebl et al., (FEMS Microbiology Letters, 53:299-303 (1989)).

Selection of the transformants took place on LBHIS agar comprising 18.5 g/l brain-heart infusion broth, 0.5 M sorbitol, 5 g/l Bacto-tryptone, 2.5 g/1 Bacto-yeast extract, 5 g/l NaCl and 18 g/l Bacto-agar, which had been supplemented with 25 mg/l kanamycin. Incubation was carried out for 2 days at 330C.

Plasmid DNA was isolated from a transformant by conventional methods (Peters-Wendisch et al., 1998, Microbiology 144, 915 -927), cleaved with the restriction endonucleases EcoRI and SalI, and the plasmid was checked by subsequent agarose gel electrophoresis. The resulting strain was called DSM5715/pZ-pfkex.

Example 20 Preparation of lysine The C. glutamicum strain DSM5715/pZ-pfkex obtained in example 4 was cultured in a nutrient medium suitable for the production of lysine and the lysine content in the culture supernatant was determined.

25 For this, the strain was first incubated on an agar plate with the corresponding antibiotic (brain-heart agar with tetracycline (5 mg/l)) for 24 hours at 33°C. Starting from this agar plate culture, a preculture was seeded (10 ml medium in a 100 ml conical flask). The complete medium Cg 30 III was used as the medium for the preculture.

o o 990179 BT AL 22 Medium Cg III NaCl Bacto-Peptone 2.5 g/l 10 g/1 10 g/1 2% (w/v) Bacto-Yeast extract Glucose (autoclaved separately) The pH was brought to pH 7.4 Kanamycin (25 mg/l) was added to this. The preculture was incubated for 16 hours at 33 0 C at 240 rpm on a shaking machine. A main culture was seeded from this preculture such that the initial OD (660nm) of the main culture was 0.05. Medium MM was used for the main culture.

990179 BT AL 23 Medium MM CSL (corn steep liquor) MOPS (morpholinopropanesulfonic acid) Glucose (autoclaved separately)

(NH

4 2 SO4

KH

2

PO

4 MgSO 4 7 H 2 0 5 g/l 20 g/l 50 g/l 25 g/l 0.1 g/l 1.0. g/1 10 mg/1 10 mg/l 0.3 mg/1 0.2 mg/1 0.1 g/l 25 g/1 CaCl 2 2 H 2 0 FeSO 4 7 H 2 0 MnSO 4

H

2 0 Biotin (sterile-filtered) 9 9 9e 9 9 .9 9 9 Thiamine HC1 (sterile-filtered) L-Leucine (sterile-filtered) CaCO 3 The CSL, MOPS and the salt solution were brought to pH 7 with aqueous ammonia and autoclaved. The sterile substrate and vitamin solutions were then added, as well as the CaCO 3 autoclaved in the dry state.

Culturing is carried out in a 10 ml volume in a 100 ml conical flask with baffles. Kanamycin (25 mg/1) was added.

Culturing was carried out at 33 0 C and 80% atmospheric humidity.

*90999 990179 BT AL 24 After 72 hours, the OD was determined at a measurement wavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich). The amount of lysine formed was determined with an amino acid analyzer from Eppendorf- BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column derivatization with ninhydrin detection.

The result of the experiment is shown in table 1.

Table 1 Strain OD(660) Lysine HC1 g/l DSM5715 7.9 13.0 DSM5715/pZ-pfkex 9.9 13.4 Figures: The following figure is attached: Figure 1: Plasmid pZ-pfkex The abbreviations used in the figure have the following meaning: Kan: Resistance gene for kanamycin Ptac: tac promoter pfk: pfk gene of C. glutamicum rrnB-T1T2: Terminator T1T2 of the rrnB gene of E.coli rep: Plasmid-coded replication origin for C. glutamicum (of pHM1519) Os S 5 S S

S

EcoRI: Cleavage site of the restriction enzyme EcoRI 990179 BT AL Sail: cleavage site of the restriction enzyme Sail 460 006S 0*0 S

'S

SOS.

*50655 S S 990179 BT AL Sequence Listing <110> Degussa-Huils AG <120> New nucleotide sequences which code for the pfk gene <130> 990179 BT <140> <141> <160> 2 <170> Patentln Ver. 2.1 <210> <211> <212> <213> <220> <221> <222> 1 1120

DNA

Corynebacterium giutamicum

CDS

(1057) <400> 1 ttgttaccga tgaccacacg ctagattttc cagttttgcc cgaccacaac tttcaggtgg taacccc atg Met 1 atc atc aca Ile Ile Thr acc cca aac ccg Thr Pro Asn Pro att gat tcc acg Ile Asp Ser Thr ctg Len tcg ctc ggc gaa Ser Leu Gly Giu ctc tcc cgt gga Leu Ser Arg Gly gtc caa cga ctt Val Gin Arg Leu tcc gtc acc gct Ser Val Thr Ala gt c Val 35 gca ggt ggt aaa Aia Gly Giy Lys ggc Giy 40 atc aat gtc gcc Ile Asn Val Ala cac gct His Ala 40 gtc ttg ctt Val Leu Leu ctc gat ccc 45 Len Asp Pro act qtt gtg Thr Val Val ggc ttt gaa acc Gly Phe Giu Thr gct gtg ttc cca Ala Val Phe Pro gcc ggc aag Aia Gly Lys ccc gtg gaa Pro Val Gin ttc gtc cca ctg Phe Val Pro Leu gt c Val 70 cgc gac atc ggc Arg Asp Ile Gly ttg Leu atc gac aag Ile Asn Lys gtc cgc acc aac Val Arg Thr Asn aca gtc acc gaa Thr Val Thr Giu ccg Pro gac ggc acc acc Asp Gly Thr Thr aag ctc aac ggc Lys Leu Asn Gly ggc gcg ccg ctc Gly Ala Pro Leu gag cag aag ctc Giu Gin Lys Len cgt Arg 115 agc ttg gaa aag Ser Len Gin Lys ctt atc gac gcg Le Ile Asp Ala ctc cgc Len Arg 125 397 445 493 ccc gaa gtc Pro Gin Val acc Thr 130 tgg gtt gtc ctg Trp Vai Val Len gcg Ala 135 ggc tcg ctg cca cca ggg gca Gly Ser Len Pro Pro Gly Ala 990179 BT AL cca gtt gac tgg Pro Val Asp Trp 145 cct gac gtt cgc Pro Asp Val Arg 160 ttg ggc gag agc Leu Gly Giu Ser 175 cca aat ggt ctg Pro Asn Gly Leu ctg gag gcg cgt Leu Glu Ala Arg 210 gcg gac gta ctg Ala Asp Val Leu 225 ggt gcc gca gga Gly Ala Ala Gly 240 act tct cca aag Thr Ser Pro Lys 255 gct ctt gca ggt Ala Leu Ala Gly gaa tct ctg ctg Glu Ser Leu Leu 290 cct ggc act acc Pro Gly Thr Thr 305 acg gtc acc caa Thr Val Thr Gln 320 tac Tyr gtg Val ttg Leu gaa Glu 195 gct Ala gtt Va1 qcg Ala att Ile ttt Phe 275 aat Asn att Ile gtc Val gcg cgt Ala Arg gct gtc Ala Val 165 gat aca Asp Thr 180 ctg ggc Leu Gly gcg caa Ala Gin aac cgt Asn Arg gtg ttg Val Leu 245 gat gtt Asp Val 260 gtt atg Val Met gcc gtg Ala Val cct cgt Pro Arg aaa gga Lys Gly 325 ctc acc gcg ttg Leu Thr Ala Leu 150 gat acc tca gac Asp Thr Ser Asp cct ggc gct gct Pro Gly Ala Ala 185 cag ctg gct aac Gin Leu Ala Asn 200 ggc gat tac gac Gly Asp Tyr Asp 215 ggc atc gaa cag Gly Ile Glu Gin 230 gtc aac gca gaa Val Asn Ala Glu gta tcc acc gtt Val Ser Thr Val 265 gca cgt tcc cag Ala Arg Ser Gin 280 tct tac ggctcg Ser Tyr Gly Ser 295 cct gac caa ctc Pro Asp Gin Leu 310 ttg aaa gaa tca Leu Lys Giu Ser atc cat Ile His 155 aag cca Lys Pro 170 ccg aac Pro Asn act gat Thr Asp gcc atc Ala Ile gtg ctt Val Leu 235 ggt gcg Gly Ala 250 gga gct Gly Ala aag aaa Lys Lys act gcg Thr Ala gcc aca Ala Thr 315 gca tgaa Ala tca Ser ctg Leu ctg Leu ggt Gly atc Ile 220 gtc Va1 tgg Trp gga Gly aca Thr gcg Ala 300 gct cgc Arg gcg Ala aag Lys 190 gag Glu gct Ala ttg Leu gct Ala tgt Cys 270 gag Glu ctt Leu gca 541 589 637 685 733 781 829 877 925 973 1021 1067 1120 Ala Gly Ala itagcgt aaataattcc tcgcttgtcc ggctggatgt cgatttcggc gactccacca cgg <210> 2 <211> 330 <212> PRT <213> Corynebacterium giutamicum <400> 2 Met Ile Ile Thr Phe Thr Pro Asn Pro Ser Ile Asp Ser Thr Leu Ser i 5 10 Leu Gly Glu Glu Leu Ser Arg Gly Ser Val Gin Arg Leu Asp Ser Val *990179 BT AL 28 25 Thr Ala Val Ala Gly Gly Lys Gly Ile Asn Val Ala His Ala Val Leu 40 Leu Ala Gly Phe Glu Thr Leu Ala Val Phe Pro Ala Gly Lys Leu Asp 55 Pro Phe Val Pro Leu Val Arg Asp Ile Gly Leu Pro Val Glu Thr Val 65 70 75 Val Ile Asn Lys Asn Val Arg Thr Asn Thr Thr Val Thr Glu Pro Asp 90 Gly Thr Thr Thr Lys Leu Asn Gly Pro Gly Ala Pro Leu Ser Glu Gin 100 105 110 Lys Leu Arg Ser Leu Glu Lys Val Leu Ile Asp Ala Leu Arg Pro Glu 115 120 125 Val Thr Trp Val Val Leu Ala Gly Ser Leu Pro Pro Gly Ala Pro Val 130 135 140 Asp Trp Tyr Ala Arg Leu Thr Ala Leu Ile His Ser Ala Arg Pro Asp 145 150 155 160 Val Arg Val Ala Val Asp Thr Ser Asp Lys Pro Leu Met Ala Leu Gly 165 170 175 Glu Ser Leu Asp Thr Pro Gly Ala Ala Pro Asn Leu Ile Lys Pro Asn 180 185 190 Gly Leu Glu Leu Gly Gin Leu Ala Asn Thr Asp Gly Glu Glu Leu Glu 195 200 205 Ala Arg Ala Ala Gin Gly Asp Tyr Asp Ala Ile Ile Ala Ala Ala Asp 210 215 220 Val Leu Val Asn Arg Gly Ile Glu Gin Val Leu Val Thr Leu Gly Ala 40 225 230 235 240 Ala Gly Ala Val Leu Val Asn Ala Glu Gly Ala Trp Thr Ala Thr Ser *oo 245 250 255 45 Pro Lys Ile Asp Val Val Ser Thr Val Gly Ala Gly Asp Cys Ala Leu 260 265 270 Ala Gly Phe Val Met Ala Arg Ser Gin Lys Lys Thr Leu Glu Glu Ser S275 280 285 Leu Leu Asn Ala Val Ser Tyr Gly Ser Thr Ala Ala Ser Leu Pro Gly 290 295 300 :*ooo Thr Thr Ile Pro Arg Pro Asp Gin Leu Ala Thr Ala Gly Ala Thr Val 55 305 310 315 320 Thr Gin Val Lys Gly Leu Lys Glu Ser Ala 325 330

Claims (18)

1. Isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence chosen from the group consisting of a) polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 2, b) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70% to the amino acid sequence of SEQ ID No. 2, c) polynucleotide which is complementary to the polynucleotides of a) or and d) polynucleotide comprising at least 15 successive bases of the polynucleotide sequence of b) or c).

2. Polynucleotide according to claim 1, wherein the polynucleotide is a DNA which is capable of replication in coryneform bacteria.

3. Polynucleotide according to claim 2 wherein the DNA is recombinant.

4. Polynucleotide according to claim 1, wherein the polynucleotide is an RNA. Polynucleotide according to claim 2 or claim 3 comprising the nucleotide sequence as shown in SEQ ID No. 1.

6. DNA according to claim 2 or claim 3 which is capable of replication, 20 comprising the nucleotide sequence shown in SEQ ID No. 1, or (ii) at least one sequence which corresponds to sequence within the range of the degeneration of the genetic code, or (iii) at least one sequence which hybridizes with the sequence complementary to 25 sequence or and optionally (iv) sense mutations of neutral function in o or 7. Polynucleotide sequence according to claim 2 or claim 3, which codes for a polypeptide which comprises the amino acid sequence in SEQ ID No. 2.

8. Isolated polynucleotide from coryneform bacteria, said polynucleotide being, substantially as hereinbefore described with reference to any one of the examples.

9. Process for the fermentative preparation of L-amino acids the following steps are carried out: a) fermentation of the L-amino acid-producing coryneform bacteria in which at least the pfk gene or nucleotide sequences which code for it are enhanced, [I:\DayLib\LIBC08643.doc:kww b) concentration of L-amino acid in the medium or in the cells of the bacteria and c) isolation of the L-amino acid. Process according to claim 9, wherein the pfk gene or nucleotide sequences which code for it are over expressed.

11. Process according to claim 9 or claim 10, wherein bacteria in which further genes of the biosynthesis pathway of the desired L-amino acid are additionally enhanced are employed.

12. Process according to claim 9 or claim 10, wherein bacteria in which the metabolic pathways which reduce the formation of L-lysine are at least partly eliminated are employed.

13. Process according to claim 9 or claim 10, wherein a strain transformed with a plasmid vector is employed, and the plasmid vector carries the nucleotide sequence which codes for the pfk gene.

14. Process according to any one of claims 9 to 13, wherein coryneform bacteria which produce L-lysine are used. Process according to claim 9 or claim 10, wherein for the preparation of lysine, bacteria in which one or more genes are chosen from the group consisting of the dapA gene which codes for dihydrodipicolinate synthase, 20 the pyc gene which codes for pyruvate carboxylase, the tpi gene which codes for trios phosphate isomerase, the gap gene which codes for glyceraldehyde 3-phosphate dehydrogenase, the pgk gene which codes for 3-phsophoglycerate kinase, the lysE gene which codes for lysine export, 25 is enhanced, at the same time are fermented.

16. Process according to claim 15, wherein said one or more genes is over "expressed or amplified.

17. Process according to claim 12, wherein for the preparation of L-lysine, bacteria in which one or more genes chosen from the group consisting of 30 the pck gene which codes for phosphoenol pyruvate carboxykinase the pgi gene which codes for glucose 6-phosphate isomerase are attenuated at the same time, are fermented.

18. Process according to any one of claims 9 to 17, wherein microorganisms of the genus Corynebacterium glutamicum are employed.

19. Process according to any one of claims 9 to 18 wherein L-lysine is produced. [I:\DayLib\LIBC]08643.doc:kww 31 Process for the fermentative preparation of L-amino acids, said process being substantially as hereinbefore described with reference to any one of the examples.

21. L-amino acids prepared by the process according to any one of claims 9 to

22. Use of a polynucleotide sequences according to any one of claims 1 to 8 as primers for the preparation of DNA genes which code for phosphofructokinase by the polymerase chain reaction.

23. Use of polynucleotide sequences according to any one of claims 1 to 8 as hybridization probes. Dated 17 November, 2000 1o Degussa-H(ls Aktiengesellschaft Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [I:\DayLib\LIBC]08643.doc:kww